NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.

Research output: Contribution to conferencePaper

Abstract

The epithelial sodium channel (ENaC) is found in a number of tissues including lung epithelia and is activated by proteolytic cleavage of its subunits by channel activating proteases (CAPs). Dysregulation of ENaC leads to increased sodium absorption which contributes to airways dehydration and thickened mucus. Impaired mucociliary clearance (MCC), characteristic of cystic fibrosis (CF) lung disease, predisposes the individual to chronic cycles of infection and inflammation, irreversible lung damage and pulmonary decline. Inhibition of CAPs therefore presents a promising target for therapy as airway rehydration through reduced ENaC activation, has the potential to restore MCC. Previous work by Reihill et al has shown that QUB-TL1, an active site-directed protease inhibitor, effectively inhibits trypsin-like enzymes and reduces ENaC-mediated sodium absorption (1). We have developed a number of other novel compounds therefore the aim of this study was to assess their ability to inhibit CAPs and to investigate downstream effects on a range of inflammatory mediators.

Four novel protease inhibitors were profiled using fluorogenic peptide substrates, before being tested for cytotoxicity by monitoring the release of lactate dehydrogenase. Downstream effects on the expression and secretion of proteinase-activated receptor 2 (PAR-2) and cytokines, IL-8 and IL-6, in CuFi cells treated with our inhibitors were investigated using qPCR and by ELISA. PAR-2 activation in a CuFi cell line was investigated through monitoring calcium mobilization.

We report NAP858, NAP1099, NAP1127 and NAP743 to be effective inhibitors of a range of CAPs. The compounds were also shown to be non-toxic to CuFi cells when treated for 24 hours when compared with vehicle alone. Initial qPCR studies indicate that treatment of CuFi cells with NAP858 and NAP1099 causes a reduction in IL-8 expression. Additionally, ELISA studies have shown that the NAP compounds are able to reduce TNFα- and LPS-stimulated IL-6 and IL-8 secretion. Calcium mobilization studies also show a reduction in PAR-2 activation after treatment with compounds.

These novel inhibitors are therefore promising follow-up compounds to QUB-TL1, presenting a potential future therapeutic option to inhibit the activation of ENaC by CAPs, as well as exerting potentially beneficial effects on downstream inflammatory signaling.

Reihill JA et al (2016) Am J Respir Crit Care Med, 194, 701-710
LanguageEnglish
Number of pages1
Publication statusPublished - 28 Oct 2017
Event10th General Meeting of the International Proteolysis Society - Banff Centre, Banff, Canada
Duration: 28 Oct 201702 Oct 2018
http://www.ips2017.org

Conference

Conference10th General Meeting of the International Proteolysis Society
CountryCanada
CityBanff
Period28/10/201702/10/2018
Internet address

Fingerprint

Cystic Fibrosis
Lung Diseases
PAR-2 Receptor
Peptide Hydrolases
Interleukin-8
Mucociliary Clearance
Protease Inhibitors
Interleukin-6
Sodium
Enzyme-Linked Immunosorbent Assay
Calcium
Epithelial Sodium Channels
Lung
Fluid Therapy
Mucus
Therapeutics
Fluorescent Dyes
Dehydration
L-Lactate Dehydrogenase
Trypsin

Cite this

Douglas, L., Ferguson, T., Reihill, J., & Martin, L. (2017). NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.. Paper presented at 10th General Meeting of the International Proteolysis Society, Banff, Canada.
Douglas, Lisa ; Ferguson, Tim ; Reihill, James ; Martin, Lorraine. / NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE. Paper presented at 10th General Meeting of the International Proteolysis Society, Banff, Canada.1 p.
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Douglas, L, Ferguson, T, Reihill, J & Martin, L 2017, 'NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.' Paper presented at 10th General Meeting of the International Proteolysis Society, Banff, Canada, 28/10/2017 - 02/10/2018, .

NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE. / Douglas, Lisa; Ferguson, Tim; Reihill, James; Martin, Lorraine.

2017. Paper presented at 10th General Meeting of the International Proteolysis Society, Banff, Canada.

Research output: Contribution to conferencePaper

TY - CONF

T1 - NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.

AU - Douglas, Lisa

AU - Ferguson, Tim

AU - Reihill, James

AU - Martin, Lorraine

PY - 2017/10/28

Y1 - 2017/10/28

N2 - The epithelial sodium channel (ENaC) is found in a number of tissues including lung epithelia and is activated by proteolytic cleavage of its subunits by channel activating proteases (CAPs). Dysregulation of ENaC leads to increased sodium absorption which contributes to airways dehydration and thickened mucus. Impaired mucociliary clearance (MCC), characteristic of cystic fibrosis (CF) lung disease, predisposes the individual to chronic cycles of infection and inflammation, irreversible lung damage and pulmonary decline. Inhibition of CAPs therefore presents a promising target for therapy as airway rehydration through reduced ENaC activation, has the potential to restore MCC. Previous work by Reihill et al has shown that QUB-TL1, an active site-directed protease inhibitor, effectively inhibits trypsin-like enzymes and reduces ENaC-mediated sodium absorption (1). We have developed a number of other novel compounds therefore the aim of this study was to assess their ability to inhibit CAPs and to investigate downstream effects on a range of inflammatory mediators. Four novel protease inhibitors were profiled using fluorogenic peptide substrates, before being tested for cytotoxicity by monitoring the release of lactate dehydrogenase. Downstream effects on the expression and secretion of proteinase-activated receptor 2 (PAR-2) and cytokines, IL-8 and IL-6, in CuFi cells treated with our inhibitors were investigated using qPCR and by ELISA. PAR-2 activation in a CuFi cell line was investigated through monitoring calcium mobilization. We report NAP858, NAP1099, NAP1127 and NAP743 to be effective inhibitors of a range of CAPs. The compounds were also shown to be non-toxic to CuFi cells when treated for 24 hours when compared with vehicle alone. Initial qPCR studies indicate that treatment of CuFi cells with NAP858 and NAP1099 causes a reduction in IL-8 expression. Additionally, ELISA studies have shown that the NAP compounds are able to reduce TNFα- and LPS-stimulated IL-6 and IL-8 secretion. Calcium mobilization studies also show a reduction in PAR-2 activation after treatment with compounds. These novel inhibitors are therefore promising follow-up compounds to QUB-TL1, presenting a potential future therapeutic option to inhibit the activation of ENaC by CAPs, as well as exerting potentially beneficial effects on downstream inflammatory signaling. Reihill JA et al (2016) Am J Respir Crit Care Med, 194, 701-710

AB - The epithelial sodium channel (ENaC) is found in a number of tissues including lung epithelia and is activated by proteolytic cleavage of its subunits by channel activating proteases (CAPs). Dysregulation of ENaC leads to increased sodium absorption which contributes to airways dehydration and thickened mucus. Impaired mucociliary clearance (MCC), characteristic of cystic fibrosis (CF) lung disease, predisposes the individual to chronic cycles of infection and inflammation, irreversible lung damage and pulmonary decline. Inhibition of CAPs therefore presents a promising target for therapy as airway rehydration through reduced ENaC activation, has the potential to restore MCC. Previous work by Reihill et al has shown that QUB-TL1, an active site-directed protease inhibitor, effectively inhibits trypsin-like enzymes and reduces ENaC-mediated sodium absorption (1). We have developed a number of other novel compounds therefore the aim of this study was to assess their ability to inhibit CAPs and to investigate downstream effects on a range of inflammatory mediators. Four novel protease inhibitors were profiled using fluorogenic peptide substrates, before being tested for cytotoxicity by monitoring the release of lactate dehydrogenase. Downstream effects on the expression and secretion of proteinase-activated receptor 2 (PAR-2) and cytokines, IL-8 and IL-6, in CuFi cells treated with our inhibitors were investigated using qPCR and by ELISA. PAR-2 activation in a CuFi cell line was investigated through monitoring calcium mobilization. We report NAP858, NAP1099, NAP1127 and NAP743 to be effective inhibitors of a range of CAPs. The compounds were also shown to be non-toxic to CuFi cells when treated for 24 hours when compared with vehicle alone. Initial qPCR studies indicate that treatment of CuFi cells with NAP858 and NAP1099 causes a reduction in IL-8 expression. Additionally, ELISA studies have shown that the NAP compounds are able to reduce TNFα- and LPS-stimulated IL-6 and IL-8 secretion. Calcium mobilization studies also show a reduction in PAR-2 activation after treatment with compounds. These novel inhibitors are therefore promising follow-up compounds to QUB-TL1, presenting a potential future therapeutic option to inhibit the activation of ENaC by CAPs, as well as exerting potentially beneficial effects on downstream inflammatory signaling. Reihill JA et al (2016) Am J Respir Crit Care Med, 194, 701-710

M3 - Paper

ER -

Douglas L, Ferguson T, Reihill J, Martin L. NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.. 2017. Paper presented at 10th General Meeting of the International Proteolysis Society, Banff, Canada.